The purification and separation of the products of biotechnological processes from the impurities and contaminants present from their production is a challenging problem in the art. Firstly, the target substances are often fragile and easily denatured biopolymers, preventing the use of conventional separation techniques employing harsh non-physiological conditions such as elevated temperatures, organic solvents or extreme pH. Further, the scale of such processes is often smaller than the conventional pharmaceutical industry, throughput and speed is often important in view of need to maintain biological activity, and the impurities and contaminants typically need to be reduced to very low levels prior to the product's intended use.
Affinity separation is based on some form of interaction between members of a specific binding pair and makes use of one of these specific interactions to force a target substance into a second phase not easily accessible to the majority of the contaminants and impurities present in the reaction mixture.
Probably the best known of these techniques is affinity chromatography in which one member of a specific binding pair is immobilised on a solid phase column. When the reaction mixture is passed through the column, only the target substance binds to the immobilised ligand, while contaminants and impurities are flushed through the column unretained. Affinity chromatography combines specificity and high enrichment factors and has been used successfully in a number of biotech downstream processes. However, it suffers from limited scale-up potential, high equipment and material costs and incompatibility with many raw feeds due to the problem of the column clogging, particularly in the early stages of product isolation.
A number of alternatives to affinity chromatography have been proposed including affinity precipitation, a term used to describe two different types of separation procedures. In so-called primary effect affinity precipitation, the affinity reaction is the direct cause for the ensuing precipitation. This method calls for at least bivalent reagents. As the affinity interaction takes place, larger and larger associates are formed which precipitate once a certain size has been surpassed which is no longer compatible with dissolution.
However, the limits of this type of process are also immediately apparent. The application range is restricted to target substances having at least two binding sites and the concentration of the target substance in a given feed must be fairly well known beforehand, since both too high or too low a concentration will result in the formation of only small associates which tend to stay in solution. In addition, there have been problems in recovering the target substance from the complexes and it can be difficult to recycle the reagents used in the process.
In secondary effect affinity precipitation, the second phase required for the separation is created by a precipitation event which is independent of the affinity interaction. This affinity interaction simply ensures the preferential transfer of the target substance into the second phase. This requires the use of bifunctional reagents, so-called affinity macroligands or AMLs comprising a reversibly soluble polymer coupled to a ligand which is a specific binding partner of the target substance. These polymers are carefully chosen for their peculiar solubility. After the target substance has bound by affinity interaction to the AML via the ligand, the entire affinity complex can be precipitated by changing a critical solution parameter such as temperature, pH, salt concentration or combinations thereof. For thermoprecipitation from aqueous solution, the LCST (lower critical solution temperature) is usually exploited, increasing the temperature of the solution to cause precipitation of the AML [1].
Previously, Freitag et al [2-5] have reported that low molar mass poly-N,N'-diethylacrylamide with molar mass distributions of less than 2 can be synthesized employing living anionic, group transfer or radical polymerization reactions in the presence of a chain transfer agent. However, when living anionic polymerization using butyl-lithium as initiator is employed, the final polymer sequences contain no reactive site for affinity ligand coupling. However, the alternative synthesis of poly-N,N'-diethylacrylamide with a carboxylic acid end group by group transfer polymerization suffers from the disadvantages that the reaction is laborious, expensive and has a very low conversion rate.
Freitag et al [4,5] have also reported that pivalyl-m-aminophenylboric acid attached via carbodiimide coupling to the carboxylic acid groups of poly-N,N'-diethylacrylamide synthesized by group transfer polymerization reaction followed by ester hydrolysis is an affinity macroligand for "Substilisin Carlsberg". In this case, the specific binding between the neutral form of phenylboric acid and the serine residue of the active enzyme centre was involved. However, it was only shown that after affinity precipitation the enzyme activity in the supernatant was reduced by 30%.
Hoffman et al [6,7] and Sakurai et al [8] have reported on the synthesis of poly-N-isopropylacrylamide with a carboxylic acid end group using 3-mercaptopropionic acid (MPA) as chain transfer agent.
In Hoffman et al [6], a poly-N-isopropylacrylamide (Mn=4,000; n=34) is disclosed which is used to make enzyme conjugates, rather than AMLs. Further, the paper indicates that this polymer would not be suitable for use in an AML as the authors observed significant loss of the conjugate after 7 cycles of precipitation-dissolution.
In the case of Hoffmann et al [7], the polymers disclosed have number molecular masses Mn greater than 4,900 (n=42).
Sakurai et al [8] have described the production of 7 poly-N-isopropylacrylamides with Mw between 1,400 and 12,300 (n=12-108) and D between 1.20 and 1.94. There is no discussion about making AMLs with these polymers and the data in the paper for polymers with low Mn (e.g. Mn=2,150; n=18; D=1.26) shows that these polymers were incompletely recovered from aqueous solution even with centrifugation, indicating that they would not be useful in the production of AMLs.
Sasakura et al [9] reported the synthesis of seven polymers with Mn between 814 and 11,480 (corresponding to n=6-91) and polydispersities between 1.18 and 2.09. In this paper, a single AML with maltose was made with one of the higher molecular mass polymers (MW=12,800; Mn=7,442, n=59) and tested in the separation of Con A. This AML had an LCST of 4.degree. C. and a NPST of 4.degree. C. The paper further discloses that it was not possible to completely recover by centrifugation polymers with lower molecular masses, e.g. showing incomplete recovery of a polymer having Mn=5,443/n=43, indicating that such polymers would not be useful in the production of AMLs.
The ideal AML comprises a reversibly precipitable polymer and a ligand capable of specific interaction with the target substance as compared to other components of the reaction mixture. Typically, as the binding of the polymer to the ligand is covalent, the base polymer needs to carry activated sites which can be reacted with the ligand. Terminal end group polymers are favoured because in this case the affinity ligand is freely accessible, and maximum activity is retained. Further, the binding sites of the ligand need to have a high degree of uniformity so that there is little variance in the interaction energy of the AML and the target substance, helping to ensure that the precipitation of both the pure AML and AML target substance complex is uniform and well defined (quantitative precipitation within a narrow temperature window) without occurrence of fractionation. Low molecular mass of the polymer may result in high ligand density. In addition to these properties, the polymer also needs to be inert with respect to ligand, target substance and impurities interaction (low non-specific interaction). For industrial use, the precipitation and resolubilization cycles should be reproducible through multiple cycles within a narrow temperature range, be substantially independent of environmental conditions and provide a high level of recovery of the AML and the target substance.
However, to date, the AMLs in the art have failed to deliver the required properties, particularly for use in the context of a robust industrial process. This means that affinity precipitation using prior art AMLs needs to be performed under highly standardized conditions, making the technique inflexible and difficult to use.